The long term objectives of this proposal are to investigate the quantitative biochemistry and physiology of bone in normal and disordered states of mineralization. We have chosen rachitic bone as a model of pathological bone disorders which express themselves as a loss in bone mass (osteoporosis), undermineralization of collagen (osteomalacia) and failure of collagen to mineralize (rachitic bone). We realize that these examples are quite different from each other in etiology and causal relationships. However, the explanation of some bone disorders on a molecular and biochemical level will provide many insights into the structure of hard tissues and possibly the molecular basis for some pathological bone disorder. Specifically we intend to determine and quantify; 1) the rate of normal bone replacement of rachitic osteoid of vitamin D deficient animals with vitamin D3, 24R, 25(OH)2D3, 2) the amount of new bone collagen synthesized that may mineralize or becomes mineralized during selected periods during the rachitic state while the animal is on a vitamin D deficient diet, 3) the molecular locations of reducible and nonreducible intermolecular structural cross-links in rachitic osteoid and normal bone collagen and correlate these data with the most frequent intermolecular distances of collagen fibrils, 4) the molecular locations of the reducible and nonreducible structural cross-links in an almost exclusive type I collagen containing noncalcified skeletal tissue such as uncalcified chick leg tendon and compare it with results from 3) above, 5) the kinetic rate of pyridinoline formation from its cross-link precursors in normal bone collagen, rachitic osteoid and noncalcified leg tendon by in vitro incubation studies in order to determine the mechanism of its formation and relationship to the three dimensional structure of collagen fibrils, and 6) the molecular location of the cross-link histidinoalanine that putatively links phophoprotein to collagen which only exists in the "mineralized" portion of normal bone and to investigate if it is present or absent in rachitic osteoid. Rachitic chicks will be repleted with vitamin D3, as well as with varying levels of 24R,25(OH)2,D3, and 1,25(OH)2D3 in concert and alone for ten days. Bone will be examined biochemically for the quality of bone and structure, (3H)Pro will be administered to vitamin D difficient 2 week-old chicks while being kept on a vitamin D deficient diet. Bone will be taken after one, two and three weeks and biochemically separated into mineralized and nonmineralized fraction (3H)Hyp in the mineralized fractions will indicate amount of calcification of D deficient state. Normal bone collagen will be separated into its mineralized and nonmineralized portion enzymatically. Each portion will be digested with trypsin and the peptides separated by gel filtration and ion-exchange chromatography. Organic phosphorous containing fractions will be analysed for Hyp, Hyl, and histidinoalanine, Amino acid and sequence analyses will give location of collagen linked phosphoprotein. Phosphoprotein and collagen structure will be modeled by computer to ascertain structural relationships.